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Calcein

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Calcein is a fluorescent dye used in various laboratory applications. It functions as a calcium indicator, allowing the detection and measurement of calcium levels in biological samples. The core purpose of Calcein is to provide a means for researchers to monitor and analyze calcium-related processes in their experiments.

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Lab products found in correlation

Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (2 mentions) 1 hydroxybenzotriazole hobt, by Merck Group (2 mentions) Victor2 multilabel counter, by PerkinElmer (2 mentions) Lsm 510, by Zeiss (2 mentions) Dm5500b, by Leica (1 mentions) Calcein, by Zeiss (1 mentions) Mitoprobe transition pore assay kit, by Thermo Fisher Scientific (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions) Facspresto system, by BD (1 mentions) Matrigel invasion chamber, by BD (1 mentions) Uncoated transwell migration chambers, by BD (1 mentions) Axiovert s100 microscope, by Zeiss (1 mentions) Axiovision 4, by Zeiss (1 mentions) Growth factor reduced matrigel, by Corning (1 mentions) Calcein, by BioLegend (1 mentions) Aria 3 sorter, by BD (1 mentions) 10 cm ultra low attachment dishes, by Corning (1 mentions) Ack buffer, by BioLegend (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by BD (1 mentions)

Close spelling variants of this product

Calcein AM (148 citations) Calcein AM fluorescent dye (19 citations) Calcein-AM dye (4 citations) Calcein acetoxymethyl ester (3 citations)

12 protocols using calcein

1

Angiogenic Assay of Thawed HUVECs

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According to modified version of a previously described protocol (15 (link)), equipment for an angiogenesis assay (BD Pharmingen, Franklin Lakes, NJ, USA) was used to determine the angiogeneic capabilities of the thawed HUVECs. In addition, the angiogenic function of the thawed HUVECs stained with calcein (BD Biosciences) was recorded using a fluorescent microscope (Leica DM5500B, Leica Microsystems, Inc.), as described previously (16 (link),17 (link)). Images from the fluorescent immunohistochemical analysis were imported into ImageJ analysis software (version 2.1; NIH, Bethesda, MD, USA; http://www.rsbweb.nih.gov/ij/).
Cai G., Lai B., Hong H., Lin P., Chen W., Zhu Z, & Chen H. (2017). Effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells. Molecular Medicine Reports, 16(1), 547-552.
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2

Measuring Mitochondrial DNA Copy Number and Volume in Hepatocytes

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Mitochondrial DNA copy number was measured by quantitative PCR as previously described(25 (link)). Primers (Integrated DNA Technologies) against part of mitochondrially-encoded NADH dehydrogenase subunit 6 (mt-Nd6) were: forward 5′-CCCAGCTACTACCATCATTCAAGT-3′ and reverse 5′-GATGGTTTGGGAGATTGGTTGATG-3′. Results shown normalized to nuclear DNA copy number. Mitochondrial volume determined by MitoTracker Red staining (Life Technologies) and divided by cell volume marked by calcein (BD Biosciences). Hepatocytes loaded with 1μM calcein and 100μM MitoTracker Red for 30min before imaging with Zeiss LSM 510 laser-scanning confocal microscope using a 63×oil lens. Z-stacks acquired of individual hepatocytes at 5μm intervals. Mitochondrial volume of a random portion of cytoplasm was determined as a fraction of cytoplasm using ImageJ’s 3D Object Counter macro (Cordelires & Jackson).
Sun Q., Loughran P., Shapiro R., Shrivastava I.H., Antoine D.J., Li T., Yan Z., Fan J., Billiar T.R, & Scott M.J. (2016). Redox-dependent regulation of hepatocyte AIM2 inflammasome activation in sterile liver injury in mice. Hepatology (Baltimore, Md.), 65(1), 253-268.
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3

Measuring Mitochondrial Permeability Transition Pore

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The mPTP opening was assessed by using MitoProbe™ Transition Pore Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction. Briefly, after treatment, culture media were removed and neurons were incubated with 1 μM calcein-AM (Life Technologies, Grand Island, NY, USA) at 37°C in the dark. Then, 1 mM CoCl2 was added and incubated with neurons for another 15 min. The fluorescence of neurons was measured with BD FACSPresto™ System (BD Biosciences, San Jose, CA, USA), and data were expressed as a percentage of mean ± standard deviation of calcein fluorescence absorbance unit (A.U) of treated neurons compared with that of untreated control neurons, which was set as 100%.
Han J., Tao W., Cui W, & Chen J. (2022). Propofol via Antioxidant Property Attenuated Hypoxia-Mediated Mitochondrial Dynamic Imbalance and Malfunction in Primary Rat Hippocampal Neurons. Oxidative Medicine and Cellular Longevity, 2022, 6298786.
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4

Tracking Transfused Murine Platelets

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A post-transfusion experiment was performed as previously described 24 (link). Briefly, washed murine platelets labeled with 5 μg/mL calcein (BD Biosciences) were incubated at RT for 1 h and were transfused into acceptor mice through the tail (1 × 108 platelets in 100 μL of MTB). Blood sampling at the tail was collected at 1 min (baseline), 6 h, 12 h, 24 h and 48 h, and total platelets were labeled with PE-anti-CD41 antibody. The percentage of calcein-labeled platelets remaining in circulation was assessed by flow cytometry.
Chen X., Wang C., Sun N., Pan S., Li R., Li X., Zhao J., Tong H., Tang Y., Han J., Qiao J., Qiu H., Wang H., Yang J, & Ikezoe T. (2021). Aurka loss in CD19+ B cells promotes megakaryocytopoiesis via IL-6/STAT3 signaling-mediated thrombopoietin production. Theranostics, 11(10), 4655-4671.
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5

Hyaluronic Acid Hydrogel Fabrication

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Hyaluronic acid (HyA, sodium salt, 1.0 MDa and 500 kDa) was generously donated by Lifecore Biomedical (Chaska, MN). Adipic dihydrazide (ADH), 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), sodium hydroxide (NaOH), hydrochloric acid (HCl) and 1-hydroxybenzotriazole (HOBt) were purchased from Aldrich (Milwaukee, WI). Dimethyl sulfoxide (DMSO), N-Acryloxysuccinimide (NAS), acetone, ethanol were obtained from Fisher Scientific (Waltham, MA). Paraformaldehyde (16% in H2O) was obtained from Electron Microscopy Sciences (Hartfield, PA). Calcein was purchased from BD Biosciences (Pasadena, CA). The MMP-degradable crosslinker peptide (CQPQGLAKC) and the 15 aminoacid adhesion peptide (CGGNGEPRGDTYRAY), bsp-RGD(15), were synthesized by American Peptide (Sunnyvale, CA). Dialysis membranes (10000 MWCO, SpectraPor Biotech CE) were purchased from Spectrum Laboratories (Rancho Dominguez, CA). All chemicals were used as received. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA). 1× Dulbecco’s phosphate buffered saline (DPBS) was purchased from Invitrogen.
Jha A.K., Tharp K.M., Ye J., Santiago-Ortiz J.L., Jackson W.M., Stahl A., Schaffer D.V., Yeghiazarians Y, & Healy K.E. (2015). Enhanced Survival and Engraftment of Transplanted Stem Cells using Growth Factor Sequestering Hydrogels. Biomaterials, 47, 1-12.
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6

Transwell Migration and Invasion Assay Protocol

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Cell migration assays were carried out using uncoated Matrigel transwell migration chambers (BD Bioscience, CA) in 24-well cell culture plates. In contrast, cell invasion assays were carried out using a Matrigel invasion chamber (BD Bioscience, CA) hydrated for at least 2 hour with 500 μl serum-free RPMI in the bottom of the well and 500 μl in the top of the chamber. After hydration of the Matrigel, DMEM in the bottom of the well was replaced with 700 μl of RPMI containing 10% FBS. Stable cells (5×104/well) were loaded in migration and invasion chambers with 500 μl of serum-free RPMI medium. In the lower chambers, 700 μl of RPMI supplemented with 10% FBS was added as a chemo-attractant. Plates were incubated for 24 or 48 hours and then stained with calcein (2 μM, BD Biosciences, CA). Cell migration and invasion were quantified using fluorescence with a VICTOR2 Multilabel Counter (Perkin Elmer) equipped with a 485/520 nm filter set.
Oh B.Y., Kim S.Y., Lee Y.S., Hong H.K., Kim T.W., Kim S.H., Lee W.Y, & Cho Y.B. (2016). Twist1-induced epithelial-mesenchymal transition according to microsatellite instability status in colon cancer cells. Oncotarget, 7(35), 57066-57076.
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7

Quantifying Leukocyte Adhesion to Endothelial Cells

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HMEC-1 cells (80–90% confluency) in 96-well plates were deprived of serum for 24h and treated with compound at indicated concentrations for 1h then incubated with IL-1ß (20 ng/mL) for 4h. Meanwhile THP-1 cells were labeled with 5µM calcein (5x106 cells/mL, BD Bioscience) for 30 min at 37°C followed by washing 3 times with PBS. THP-1 (2.5x105 cells/well) were then added for 30 min, unbound cells were washed off 3 times in PBS. Adhesion of calcein-labeled THP-1 to the endothelium layer was determined in a fluorescent plate reader at excitation 485 nm and emission 530 nm.
Yeh M.C., Wu B.J., Li Y., Elahy M., Prado-Lourenco L., Sockler J., Lau H., Day R.O, & Khachigian L.M. (2021). BT2 Suppresses Human Monocytic-Endothelial Cell Adhesion, Bone Erosion and Inflammation. Journal of Inflammation Research, 14, 1019-1028.
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8

Cell Migration and Invasion Assay

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Cell migration and invasion assays were carried out using uncoated trans-well migration chambers (BD Bioscience, San Jose, CA, USA) in 24-well cell culture plates. Cells (5×104 /well) were loaded into the migration and invasion chambers in serum-free RPMI media. The lower chambers contained RPMI supplemented with 10% FBS as a chemoattractant. Plates were incubated for 24 or 48 hours. Cells were then stained with calcein (2 μM, BD Biosciences, San Jose, CA, USA) or hematoxylin-eosin and mounted. Cell migration and invasion was quantified by fluorescence measurements with a VICTOR2 Multilabel Counter (Perkin Elmer, Boston, MA, USA) equipped with a 485/520 nm filter set.
Lee Y.S., Kim S.Y., Song S.J., Hong H.K., Lee Y., Oh B.Y., Lee W.Y, & Cho Y.B. (2016). Crosstalk between CCL7 and CCR3 promotes metastasis of colon cancer cells via ERK-JNK signaling pathways. Oncotarget, 7(24), 36842-36853.
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9

Angiogenic Potential of Epigenetically Modulated ECFCs

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ECFCs, pretreated with epigenetic drugs or vehicle as indicated, were seeded into 96-well plates previously coated with 50 μL of phenol-red-free, growth factor reduced Matrigel (Corning) at a density of 5,000 cells/well in duplicates. After 24 hr, cells were stained with 5 μg/mL calcein (BD Bioscience) for 5 min at 37°C and photographed using an Axiovert S100 microscope (Zeiss) and AxioVision 4.6 software at 2.5× magnification. Total tube length was quantified using AngioQuant software as described (Fraineau et al., 2012 (link)).
Fraineau S., Palii C.G., McNeill B., Ritso M., Shelley W.C., Prasain N., Chu A., Vion E., Rieck K., Nilufar S., Perkins T.J., Rudnicki M.A., Allan D.S., Yoder M.C., Suuronen E.J, & Brand M. (2017). Epigenetic Activation of Pro-angiogenic Signaling Pathways in Human Endothelial Progenitors Increases Vasculogenesis. Stem Cell Reports, 9(5), 1573-1587.
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10

Isolation of TNBC Immune Cells

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Triple negative breast cancer immune cells were processed as previously described [12 (link)]. Briefly, fresh TNBC core biopsy tumor specimens were received in MACS Tissue Storage Solution (Cat #130-100-008) on ice and minced in 10 cm ultra-low attachment dishes (Cat # 3262, Corning), and digested with Rat tail collagenase IV (Cat # 17104019, Life Technologies) for 30 min. Red blood cells were lysed with ACK buffer (Cat # 420301, BioLegend) and single cells were obtained and stained with DAPI (Cat # 422801, Biolegend) and Calcein (Cat # 425201, Biolegend). The BD Aria III sorter was used with a 100 μm nozzle to sort live (DAPI- Calcein+) single cells.
Deng J., Thennavan A., Shah S., Bagdatlioglu E., Klar N., Heguy A., Marier C., Meyn P., Zhang Y., Labbe K., Almonte C., Krogsgaard M., Perou C.M., Wong K.K, & Adams S. (2020). Serial single-cell profiling analysis of metastatic TNBC during Nab-paclitaxel and pembrolizumab treatment. Breast cancer research and treatment, 185(1), 85-94.
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